α-Thrombin plays an important role in hemostasis and hence is a highly useful protein for use as a hemostatic. Currently, α-thrombin is utilized in more than 1 000 000 patients in the USA each year [1]. The main source of α-thrombin is pooled human plasma collected from donors. The plasma is treated by complicated filtration and separation, however, no procedure is completely effective against viral particles derived from human blood [2].
Alternatively, recombinant α-thrombin can be produced that is devoid of the risks of viral particles. Thrombin is synthesized in the form of prothrombin which is one of proteolytic enzymes which are normally synthesized in their inactive form, known as proenzymes or zymogens, which can be cleaved by factor Xa or by ecarin, a protease found in snake poison, [3] at two places, Arg-Thr and Arg-Ile bonds. Cleavage at the first factor Xa site results in prethrombin-2, an inactive single-chain precursor that has the same size as α-thrombin. Activation of prethrombin-2 to α-thrombin occurs through internal rearrangement of the initial peptide chain upon cleavage of Arg-Ile bond with factor Xa or by ecarin [4]. The cleaved shorter peptide section does not leave the α-thrombin macromolecule but stays linked with the longer peptide sequence via an S—S bond.
Currently, it is impossible to directly express active recombinant α-thrombin from the gene fragment corresponding to prothrombin because the resulting protein will be always inactive prethrombin-2. The preparation of recombinant prethrombin-2 and its activation to α-thrombin by ecarin, has been described in the literature [5]. The main drawback of this procedure is the necessity to employ extremely dangerous ecarin, the primary reagent in the venom of the snake Echis carinatus [6] to activate α-thrombin. The removal of ecarin, significantly diminishes the yield and increases costs of α-thrombin preparation. Still there is no 100% assurance that recombinant α-thrombin is not contaminated with ecarin. Factor Xa can be used instead of lethal ecarin but it requires for its optimal operation a complex with factor V, platelet phospholipids and calcium [7], that also must be separated from α-thrombin.
On the other hand, enzymes, such us, α-thrombin, have been commonly used in different bioanalytical assays for detection and amplification of signal. They are employed in quantification of glucose [8], H2O2 [9], pesticides [10], cholesterol [11], and ethanol [12], and are the basis for ELISAs (enzyme linked immunosorbent assays) [13]. Enzyme amplification techniques [14] have been used to improve the sensitivity of several bioanalytical assays. In this sense, the state of the art discloses an assay including a double amplification cascade in which ecarin, converted prothrombin to α-thrombin to digest an artificial fluorogenic substrate [15]. As mentioned above, prothrombin is one of proteolytic enzymes which are normally synthesized in their inactive form, known as proenzymes or zymogens [16]. When the product of the proenzyme cleavage reaction catalyzes the same reaction, the process is called an autocatalytic activation [17]. Some examples of natural autocatalytic enzymes are trypsinogen, pepsinogen, or the blood coagulation factor XII [18]. The autocatalytic behavior of these enzymes could be applied for analytical purposes. Moreover, tests for α-thrombin activity are used to evaluate the rate of blood coagulation, therefore it is important to develop sensitive methods to monitor its activity.
The signal obtained by a low concentration of an enzyme can be considerably amplified by means of an autocatalytic reaction. The use of zymogens in bioanalysis is limited since all known natural autocatalytic proenzymes are unstable in vitro and their preparations always contain traces of corresponding active enzymes [19]. To the best of our knowledge, there are still no commercially available kits based on natural autocatalytic proenzymes. Previously, the inventors used the self-replicating DNA machines based on endonucleases to create signal amplification networks [20, 21]. Unfortunately, DNA machines are not stable in body liquids and can not be applied for the analysis of blood derived samples.
In spite of considerable research into the signal amplification in bioassays, specifically, using self-replicating enzymes and also the preparation and production of these enzymes, more studies are still needed. Moreover, it has not yet been found a proenzyme stable in vitro without traces of corresponding active enzymes and also with autocatalytic activity, particularly these is not disclosures in the prior art about a recombinant prethrombin-2, with autocatalytic features, which show stability in vitro, without traces of α-thrombin.